Induction of exon skipping in eukaryotic cells

ABSTRACT

The present invention provides a method for at least in part decreasing the production of an aberrant protein in a cell, the cell comprising pre-mRNA comprising exons coding for the protein, by inducing so-call exon skipping in the cell. Exon-skipping results in mature mRNA that does not contain the skipped exon, which leads to an altered product of the exon codes for amino acids. Exon skipping is performed by providing a cell with an agent capable of specifically inhibiting an exon inclusion signal, for instance, an exon recognition sequence, of the exon. The exon inclusion signal can be interfered with by a nucleic acid comprising complementarity to a part of the exon. The nucleic acid, which is also herewith provided, can be used for the preparation of a medicament, for instance, for the treatment of an inherited disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 10/395,031, filed Mar. 21, 2003, now U.S. Pat. No. ______,which is a continuation of International Application PCT/NL01/00697,filed Sep. 21, 2001, designating the United States, published in EnglishMar. 28, 2002, as WO 02/024906 A1 and subsequently published withcorrections Jan. 23, 2003, as WO 02/024906 C2, the contents of theentirety of each of which are hereby incorporated herein by thisreference.

TECHNICAL FIELD

The invention relates to the field of gene therapy.

BACKGROUND

Given the rapid advances of human genome research, professionals and thepublic expect that the near future will bring us, in addition tounderstanding of disease mechanisms and refined and reliablediagnostics, therapies for many devastating genetic diseases.

While it is hoped that for some (e.g., metabolic) diseases, the improvedinsights will bring easily administrable small-molecule therapies, it islikely that in most cases one or another form of gene therapy willultimately be required, i.e., the correction, addition or replacement ofthe defective gene product.

In the past few years, research and development in this field havehighlighted several technical difficulties which need to be overcome,e.g., related to the large size of many genes involved in geneticdisease (limiting the choice of suitable systems to administer thetherapeutic gene), the accessibility of the tissue in which thetherapeutic gene should function (requiring the design of specifictargeting techniques, either physically, by restricted injection, orbiologically, by developing systems with tissue-specific affinities) andthe safety to the patient of the administration system. These problemsare to some extent interrelated, and it can be generally concluded thatthe smaller the therapeutic agent is, the easier it will become todevelop efficient, targetable and safe administration systems.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses this problem by inducing so-calledexon-skipping in cells. Exon-skipping results in mature mRNA that doesnot contain the skipped exon and thus, when the exon codes for aminoacids, can lead to the expression of an altered product. Technology forexon-skipping is currently directed toward the use of so-called“Anti-sense Oligonucleotides” (AONs).

Much of this work is done in the mdx mouse model for Duchenne musculardystrophy (DMD). The mdx mouse, which carries a nonsense mutation inexon 23 of the dystrophin gene, has been used as an animal model ofDuchenne muscular dystrophy. Despite the mdx mutation, which shouldpreclude the synthesis of a functional dystrophin protein, rare,naturally occurring dystrophin-positive fibers have been observed in mdxmuscle tissue. These dystrophin-positive fibers are thought to havearisen from an apparently naturally occurring exon-skipping mechanism,either due to somatic mutations or through alternative splicing.

AONs directed to, respectively, the 3′ and 5′ splice sites of introns 22and 23 in dystrophin pre-mRNA have been shown to interfere with factorsnormally involved in removal of intron 23 so that exon 23 was alsoremoved from the mRNA (Wilton, 1999). In a similar study, Dunckley etal. (1998) showed that exon skipping using AONs directed to3′ and 5′splice sites can have unexpected results. They observed skipping of notonly exon 23 but also of exons 24-29, thus resulting in an mRNAcontaining an exon 22-exon 30 junction.

The underlying mechanism for the appearance of the unexpected 22-30splicing variant is not known. It could be due to the fact that splicesites contain consensus sequences leading to promiscuous hybridizationof the oligos used to direct the exon skipping. Hybridization of theoligos to other splice sites than the sites of the exon to be skipped ofcourse could easily interfere with the accuracy of the splicing process.On the other hand, the accuracy could be lacking due to the fact thattwo oligos (for the 5′ and the 3′ splice site) need to be used. Pre-mRNAcontaining one but not the other oligo could be prone to unexpectedsplicing variants.

To overcome these and other problems, the present invention provides amethod for directing splicing of a pre-mRNA in a system capable ofperforming a splicing operation comprising contacting the pre-mRNA inthe system with an agent capable of specifically inhibiting an exoninclusion signal of at least one exon in the pre-mRNA, the methodfurther comprising allowing splicing of the pre-mRNA. Interfering withan exon inclusion signal (EIS) has the advantage that such elements arelocated within the exon. By providing an antisense oligo for theinterior of the exon to be skipped, it is possible to interfere with theexon inclusion signal, thereby effectively masking the exon from thesplicing apparatus. The failure of the splicing apparatus to recognizethe exon to be skipped thus leads to exclusion of the exon from thefinal mRNA.

The present invention does not interfere directly with the enzymaticprocess of the splicing machinery (the joining of the exons). It isthought that this allows the method to be more robust and reliable. Itis thought that an EIS is a particular structure of an exon that allowssplice acceptor and donor to assume a particular spatial conformation.In this concept, it is the particular spatial conformation that enablesthe splicing machinery to recognize the exon. However, the invention iscertainly not limited to this model.

It has been found that agents capable of binding to an exon can inhibitan EIS. Agents may specifically contact the exon at any point and stillbe able to specifically inhibit the EIS. The mRNA may be useful initself. For instance, production of an undesired protein can be at leastin part reduced by inhibiting inclusion of a required exon into themRNA. A preferred method of the invention further comprises allowingtranslation of mRNA produced from splicing of the pre-mRNA. Preferably,the mRNA encodes a functional protein. In a preferred embodiment, theprotein comprises two or more domains, wherein at least one of thedomains is encoded by the mRNA as a result of skipping of at least partof an exon in the pre-mRNA.

Exon skipping will typically, though not necessarily, be of relevancefor proteins in the wild-type configuration, having at least twofunctional domains that each performs a function, wherein the domainsare generated from distinct parts of the primary amino acid sequence.Examples are, for instance, transcription factors. Typically, thesefactors comprise a DNA binding domain and a domain that interacts withother proteins in the cell. Skipping of an exon that encodes a part ofthe primary amino acid sequence that lies between these two domains canlead to a shorter protein that comprises the same function, at least inpart. Thus, detrimental mutations in this intermediary region (forinstance, frame-shift or stop mutations) can be at least in partrepaired by inducing exon skipping to allow synthesis of the shorter(partly) functional protein.

Using a method of the invention, it is also possible to induce partialskipping of the exon. In this embodiment, the contacting results inactivation of a cryptic splice site in a contacted exon. This embodimentbroadens the potential for manipulation of the pre-mRNA leading to afunctional protein. Preferably, the system comprises a cell. Preferably,the cell is cultured in vitro or in the organism in vivo. Typically,though not necessarily, the organism comprises a human or a mouse.

In a preferred embodiment, the invention provides a method for at leastin part decreasing the production of an aberrant protein in a cell, thecell comprising pre-mRNA comprising exons coding for the protein, themethod comprising providing the cell with an agent capable ofspecifically inhibiting an exon inclusion signal of at least one of theexons, the method further comprising allowing translation of mRNAproduced from splicing of the pre-mRNA.

Any agent capable of specifically inhibiting an exon exclusion signalcan be used for the present invention. Preferably, the agent comprises anucleic acid or a functional equivalent thereof Preferably, but notnecessarily, the nucleic acid is in single-stranded form. Peptidenucleic acid and other molecules comprising the same nucleic acidbinding characteristics in kind, but not necessarily in amount, aresuitable equivalents. Nucleic acid or an equivalent may comprisemodifications to provide additional functionality. For instance,2′-O-methyl oligoribonucleotides can be used. These ribonucleotides aremore resistant to RNAse action than conventional oligonucleotides.

In a preferred embodiment of the invention, the exon inclusion signal isinterfered with by an antisense nucleic acid directed to an exonrecognition sequence (ERS). These sequences are relatively purine-richand can be distinguished by scrutinizing the sequence information of theexon to be skipped (Tanaka et al., 1994, Mol. Cell. Biol. 14, p.1347-1354). Exon recognition sequences are thought to aid inclusion intomRNA of so-called weak exons (Achsel et al., 1996, J. Biochem. 120, p.53-60). These weak exons comprise, for instance, 5′ and or 3′ splicesites that are less efficiently recognized by the splicing machinery. Inthe present invention, it has been found that exon skipping can also beinduced in so-called strong exons, i.e., exons which are normallyefficiently recognized by the splicing machinery of the cell. From anygiven sequence, it is (almost) always possible to predict whether thesequence comprises putative exons and to determine whether these exonsare strong or weak. Several algorithms for determining the strength ofan exon exist. A useful algorithm can be found on the NetGene2 splicesite prediction server (Brunak, et al., 1991, J. Mol. Biol. 220, p.49-65). Exon skipping by a means of the invention can be induced in(almost) every exon, independent of whether the exon is a weak exon or astrong exon and also independent of whether the exon comprises an ERS.In a preferred embodiment, an exon that is targeted for skipping is astrong exon. In another preferred embodiment, an exon targeted forskipping does not comprise an ERS.

Methods of the invention can be used in many ways. In one embodiment, amethod of the invention is used to at least in part decrease theproduction of an aberrant protein. Such proteins can, for instance, beonco-proteins or viral proteins. In many tumors, not only the presenceof an onco-protein but also its relative level of expression has beenassociated with the phenotype of the tumor cell. Similarly, not only thepresence of viral proteins but also the amount of viral protein in acell determines the virulence of a particular virus. Moreover, forefficient multiplication and spread of a virus, the timing of expressionin the life cycle and the balance in the amount of certain viralproteins in a cell determines whether viruses are efficiently orinefficiently produced. Using a method of the invention, it is possibleto lower the amount of aberrant protein in a cell such that, forinstance, a tumor cell becomes less tumorigenic (metastatic) and/or avirus-infected cell produces less virus.

In a preferred embodiment, a method of the invention is used to modifythe aberrant protein into a functional protein. In one embodiment, thefunctional protein is capable of performing a function of a proteinnormally present in a cell but absent in the cells to be treated. Veryoften, even partial restoration of function results in significantlyimproved performance of the cell thus treated. Due to the betterperformance, such cells can also have a selective advantage overunmodified cells, thus aiding the efficacy of the treatment.

This aspect of the invention is particularly suited for the restorationof expression of defective genes. This is achieved by causing thespecific skipping of targeted exons, thus bypassing or correctingdeleterious mutations (typically stop-mutations or frame-shifting pointmutations, single- or multi-exon deletions or insertions leading totranslation termination).

Compared to gene-introduction strategies, this novel form ofsplice-modulation gene therapy requires the administration of muchsmaller therapeutic reagents, typically, but not limited to, 14-40nucleotides. In a preferred embodiment, molecules of 14-25 nucleotidesare used since these molecules are easier to produce and enter the cellmore effectively. The methods of the invention allow much moreflexibility in the subsequent design of effective and safeadministration systems. An important additional advantage of this aspectof the invention is that it restores (at least some of) the activity ofthe endogenous gene, which still possesses most or all of itsgene-regulatory circuitry, thus ensuring proper expression levels andthe synthesis of tissue-specific isoforms.

This aspect of the invention can in principle be applied to any geneticdisease or genetic predisposition to disease in which targeted skippingof specific exons would restore the translational reading frame whenthis has been disrupted by the original mutation, provided thattranslation of an internally slightly shorter protein is still fully orpartly functional. Preferred embodiments for which this application canbe of therapeutic value are: predisposition to second hit mutations intumor suppressor genes, e.g., those involved in breast cancer, coloncancer, tuberous sclerosis, neurofibromatosis etc., where (partial)restoration of activity would preclude the manifestation of nullosomy bysecond hit mutations and thus would protect against tumorigenesis.Another preferred embodiment involves the (partial) restoration ofdefective gene products which have a direct disease causing effect,e.g., hemophilia A (clotting factor VIII deficiency), some forms ofcongenital hypothyroidism (due to thyroglobulin synthesis deficiency)and Duchenne muscular dystrophy (DMD), in which frame-shiftingdeletions, duplications and stop mutations in the X-linked dystrophingene cause severe, progressive muscle degradation. DMD is typicallylethal in late adolescence or early adulthood, while non-frame-shiftingdeletions or duplications in the same gene cause the much milder Beckermuscular dystrophy (BMD), compatible with a life expectancy between35-40 years to normal. In the embodiment as applied to DMD, the presentinvention enables exon skipping to extend an existing deletion (or alterthe mRNA product of an existing duplication) by as many adjacent exonsas required to restore the reading frame and generate an internallyslightly shortened, but still functional, protein. Based on the muchmilder clinical symptoms of BMD patients with the equivalent of thisinduced deletion, the disease in the DMD patients would have a muchmilder course after AON-therapy.

Many different mutations in the dystrophin gene can lead to adysfunctional protein. (For a comprehensive inventory see www.dmd.nl,the internationally accepted database for DMD and related disorders.)The precise exon to be skipped to generate a functional dystrophinprotein varies from mutation to mutation. Table 1 comprises anon-limiting list of exons that can be skipped and lists for thementioned exons some of the more frequently occurring dystrophin genemutations that have been observed in humans and that can be treated witha method of the invention. Skipping of the mentioned exon leads to amutant dystrophin protein comprising at least the functionality of aBecker mutant. Thus, in one embodiment, the invention provides a methodof the invention wherein the exon inclusion signal is present in exonnumbers 2, 8, 19, 29, 43, 44, 45, 46, 50, 51, 52 or 53 of the humandystrophin gene. The occurrence of certain deletion/insertion variationsis more frequent than others. In the present invention, it was foundthat by inducing skipping of exon 46 with a means or a method of theinvention, approximately 7% of DMD-deletion containing patients can betreated, resulting in the patients to comprise dystrophin-positivemuscle fibers. By inducing skipping of exon 51, approximately 15% ofDMD-deletion containing patients can be treated with a means or methodof the invention. Such treatment will result in the patient having atleast some dystrophin-positive fibers. Thus, with either skipping ofexon 46 or 51 using a method of the invention, approximately 22% of thepatients containing a deletion in the dystrophin gene can be treated.Thus, in a preferred embodiment of the invention, the exon exclusionsignal is present in exon 46 or exon 51. In a particularly preferredembodiment, the agent comprises a nucleic acid sequence according tohAON#4, hAON#6, hAON#8, hAON#9, hAON#11 and/or one or more of hAON#21-30or a functional part, derivative and/or analogue of the hAON. Afunctional part, derivative and/or analogue of the hAON comprises thesame exon skipping activity in kind, but not necessarily in amount, in amethod of the invention.

TABLE 1 Therapeutic for Exon to be DMD-deletions Frequency in skipped(exons) www.dmd.nl (%) 2 3-7 2 8 3-7 4 4-7 5-7 6-7 43 44 5 44-47 4435-43 8 45 45-54 45 18-44 13 46-47 44 46-48 46-49 46-51 46-53 46 45 7 5051 5 51-55 51 50 15 45-50 48-50 49-50 52 52-63 52 51 3 53 53-55 53 45-529 48-52 49-52 50-52 52

It can be advantageous to induce exon skipping of more than one exon inthe pre-mRNA. For instance, considering the wide variety of mutationsand the fixed nature of exon lengths and amino acid sequence flankingsuch mutations, the situation can occur that for restoration of functionmore than one exon needs to be skipped. A preferred but non-limitingexample of such a case in the DMD deletion database is a 46-50 deletion.Patients comprising a 46-50 deletion do not produce functionaldystrophin. However, an at least partially functional dystrophin can begenerated by inducing skipping of both exon 45 and exon 51. Anotherpreferred but non-limiting example is patients comprising a duplicationof exon 2. By providing one agent capable of inhibiting an EIS of exon2, it is possible to partly skip either one or both exons 2, therebyregenerating the wild-type protein next to the truncated or double exon2 skipped protein. Another preferred but non-limiting example is theskipping of exons 45 through 50. This generates an in-frame Becker-likevariant. This Becker-like variant can be generated to cure any mutationlocalized in exons 45, 46, 47, 48, 49, and/or 50 or combinationsthereof. In one aspect, the invention therefore provides a method of theinvention further comprising providing the cell with another agentcapable of inhibiting an exon inclusion signal in another exon of thepre-mRNA. Of course, it is completely within the scope of the inventionto use two or more agents for the induction of exon skipping in pre-mRNAof two or more different genes.

In another aspect, the invention provides a method for selecting thesuitable agents for splice-therapy and their validation as specificexon-skipping agents in pilot experiments. A method is provided fordetermining whether an agent is capable of specifically inhibiting anexon inclusion signal of an exon, comprising providing a cell having apre-mRNA containing the exon with the agent, culturing the cell to allowthe formation of an mRNA from the pre-mRNA and determining whether theexon is absent the mRNA. In a preferred embodiment, the agent comprisesa nucleic acid or a functional equivalent thereof, the nucleic acidcomprising complementarity to a part of the exon. Agents capable ofinducing specific exon skipping can be identified with a method of theinvention. It is possible to include a prescreen for agents by firstidentifying whether the agent is capable of binding with a relativelyhigh affinity to an exon containing nucleic acid, preferably RNA. Tothis end, a method for determining whether an agent is capable ofspecifically inhibiting an exon inclusion signal of an exon is provided,further comprising first determining in vitro the relative bindingaffinity of the nucleic acid or functional equivalent thereof to an RNAmolecule comprising the exon.

In yet another aspect, an agent is provided that is obtainable by amethod of the invention. In a preferred embodiment, the agent comprisesa nucleic acid or a functional equivalent thereof Preferably the agent,when used to induce exon skipping in a cell, is capable of at least inpart reducing the amount of aberrant protein in the cell. Morepreferably, the exon skipping results in an mRNA encoding a protein thatis capable of performing a function in the cell. In a particularlypreferred embodiment, the pre-mRNA is derived from a dystrophin gene.Preferably, the functional protein comprises a mutant or normaldystrophin protein. Preferably, the mutant dystrophin protein comprisesat least the functionality of a dystrophin protein in a Becker patient.In a particularly preferred embodiment, the agent comprises the nucleicacid sequence of hAON#4, hAON#6, hAON#8, hAON#9, hAON#11 and/or one ormore of hAON#21-30 or a functional part, derivative and/or analogue ofthe hAON. A functional part, derivative and/or analogue of the hAONcomprises the same exon skipping activity in kind, but not necessarilyin amount, in a method of the invention.

The art describes many ways to deliver agents to cells. Particularly,nucleic acid delivery methods have been widely developed. The artisan iswell capable of determining whether a method of delivery is suitable forperforming the present invention. In a non-limiting example, the methodincludes the packaging of an agent of the invention into liposomes, theliposomes being provided to cells comprising a target pre-mRNA.Liposomes are particularly suited for delivery of nucleic acid to cells.Antisense molecules capable of inducing exon skipping can be produced ina cell upon delivery of nucleic acid containing a transcription unit toproduce antisense RNA. Non-limiting examples of suitable transcriptionunits are small nuclear RNA (SNRP) or tRNA transcription units. Theinvention, therefore, further provides a nucleic acid delivery vehiclecomprising a nucleic acid or functional equivalent thereof of theinvention capable of inhibiting an exon inclusion signal. In oneembodiment, the delivery vehicle is capable of expressing the nucleicacid of the invention. Of course, in case, for instance, single-strandedviruses are used as a vehicle, it is entirely within the scope of theinvention when such a virus comprises only the antisense sequence of anagent of the invention. In another embodiment of single strand viruses,AONs of the invention are encoded by small nuclear RNA or tRNAtranscription units on viral nucleic encapsulated by the virus asvehicle. A preferred single-stranded virus is adeno-associated virus.

In yet another embodiment, the invention provides the use of a nucleicacid or a nucleic acid delivery vehicle of the invention for thepreparation of a medicament. In a preferred embodiment, the medicamentis used for the treatment of an inherited disease. More preferably, themedicament is used for the treatment of Duchenne Muscular Dystrophy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Deletion of exon 45 is one of the most frequent DMD-mutations.Due to this deletion, exon 44 is spliced to exon 46, the translationalreading frame is interrupted, and a stop codon is created in exon 46leading to a dystrophin deficiency. Our aim is to artificially inducethe skipping of an additional exon, exon 46, in order to reestablish thereading frame and restore the synthesis of a slightly shorter, butlargely functional, dystrophin protein as found in the much milderaffected Becker muscular dystrophy patients affected by a deletion ofboth exons 45 and 46.

FIG. 2. Exon 46 contains a purine-rich region that is hypothesized tohave a potential role in the regulation of its splicing in the pre-mRNA.A series of overlapping 2′O-methyl phosphorothioate antisenseoligoribonucleotides (AONs) was designed directed at this purine-richregion in mouse dystrophin exon 46. The AONs differ both in length andsequence. The chemical modifications render the AONs resistant toendonucleases and RNaseH inside the muscle cells. To determine thetransfection efficiency in our in vitro studies, the AONs contained a 5′fluorescein group which allowed identification of AON-positive cells.

FIG. 3. To determine the binding affinity of the different AONs to thetarget exon 46 RNA, we performed gel mobility shift assays. In thisfigure, the five mAONs (mAON#4, 6, 8, 9, and 11) with highest affinityfor the target RNA are shown. Upon binding of the AONs to the RNA, acomplex is formed that exhibits a retarded gel mobility as can bedetermined by the band shift. The binding of the AONs to the target wassequence-specific. A random mAON, i.e. not specific for exon 46, did notgenerate a band shift.

FIGS. 4A and 4B. The mouse- and human-specific AONs which showed thehighest binding affinity in the gel mobility shift assays weretransfected into mouse and human myotube cultures.

FIG. 4A. RT-PCR analysis showed a truncated product, of which the sizecorresponded to exon 45 directly spliced to exon 47, in the mouse cellcultures upon transfection with the different mAONs#4, 6, 9, and 11. Noexon 46 skipping was detected following transfection with a random AON.

FIG. 4B. RT-PCR analysis in the human muscle cell cultures derived fromone unaffected individual (C) and two unrelated DMD patients (P1 and P2)revealed truncated products upon transfection with hAON#4 and hAON#8. Inthe control, this product corresponded to exon 45 spliced to exon 47,while in the patients, the fragment size corresponded to exon 44 splicedto exon 47. No exon 46 skipping was detected in the non-transfected cellcultures or following transfection with a random hAON. Highest exon 46skipping efficiencies were obtained with hAON#8.

FIG. 5. Sequence data from the RT-PCR products obtained from patientDL279.1 (corresponding to P1 in FIG. 4), which confirmed the deletion ofexon 45 in this patient (upper panel), and the additional skipping ofexon 46 following transfection with hAON#8 (lower panel). The skippingof exon 46 was specific, and exon 44 was exactly spliced to exon 47,which reestablishes the translational reading frame.

FIG. 6. Immunohistochemical analysis of the muscle cell culture frompatient DL279.1 upon transfection with hAON#8. Cells were subject to twodifferent dystrophin antibodies raised against different regions of theprotein, located proximally (ManDys-1, ex. 31-32) and distally (Dys-2,ex. 77-79) from the targeted exon 46. The lower panel shows the absenceof a dystrophin protein in the myotubes, whereas the hAON#8-inducedskipping of exon 46 clearly restored the synthesis of a dystrophinprotein as detected by both antibodies (upper panel).

FIG. 7A. RT-PCR analysis of RNA isolated from human control muscle cellcultures treated with hAON#23, #24, #27, #28, or #29. An additionalaberrant splicing product was obtained in cells treated with hAON#28 and#29. Sequence analysis revealed the utilization of an in-frame crypticsplice site within exon 51 that is used at a low frequency upon AONtreatment. The product generated included a partial exon 51, which alsohad a restored reading frame, thereby confirming further the therapeuticvalue.

FIG. 7B. A truncated product, with a size corresponding to exon 50spliced to exon 52, was detected in cells treated with hAON#23 and #28.Sequence analysis of these products confirmed the precise skipping ofexon 51.

FIG. 8A. Gel mobility shift assays were performed to determine thebinding affinity of the different h29AON#'s for the exon 29 target RNA.When compared to non-hybridized RNA (none), h29AON#1, #2, #4, #6, #9,#10, and #11 generated complexes with lower gel mobilities, indicatingtheir binding to the RNA. A random AON derived from dystrophin exon 19did not generate a complex.

FIG. 8B. RT-PCR analysis of RNA isolated from human control muscle cellcultures treated with h29AON#1, #2, #4, #6, #9, #10, or #11 revealed atruncated product of which the size corresponded to exon 28 spliced toexon 30. These results indicate that exon 29 can specifically be skippedusing AONs directed to sequences either within (h29AON#1, #2, #4, or #6)or outside (h29AON#9, #10, or #11) the hypothesized ERS in exon 29. Anadditional aberrant splicing product was observed that resulted fromskipping of both exon 28 and exon 29 (confirmed by sequence data notshown). Although this product was also present in non-treated cells,suggesting that this alternative skipping event may occur naturally, itwas enhanced by the AON-treatment. AON 19, derived from dystrophin exon19, did not induce exon 29 skipping.

FIG. 8C. The specific skipping of exon 29 was confirmed by sequence datafrom the truncated RT-PCR fragments. Shown here is the sequence obtainedfrom the exon 29 skipping product in cells treated with h29AON#1.

FIG. 9A. RT-PCR analysis of RNA isolated from mouse gastrocnemiusmuscles two days post-injection of 5, 10, or 20 ng of either mAON#4, #6,or #11. Truncated products, with a size corresponding to exon 45 splicedto exon 47, were detected in all treated muscles. The samples -RT, -RNA,AD-1, and AD-2 were analyzed as negative controls for the RT-PCRreactions.

FIG. 9B. Sequence analysis of the truncated products generated by mAON#4and #6 (and #11, not shown) confirmed the precise skipping of exon 46.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES Example 1

Since exon 45 is one of the most frequently deleted exons in DMD, weinitially aimed at inducing the specific skipping of exon 46 (FIG. 1).This would produce the shorter, largely functional dystrophin found inBMD patients carrying a deletion of exons 45 and 46. The system wasinitially set up for modulation of dystrophin pre-mRNA splicing of themouse dystrophin gene. We later aimed for the human dystrophin gene withthe intention to restore the translational reading frame and dystrophinsynthesis in muscle cells from DMD patients affected by a deletion ofexon 45.

Design of mAONs and hAONs

A series of mouse- and human-specific AONs (mAONs and hAONs) wasdesigned, directed at an internal part of exon 46 that contains astretch of purine-rich sequences and is hypothesized to have a putativeregulatory role in the splicing process of exon 46 (FIG. 2). For theinitial screening of the AONs in the gel mobility shift assays (seebelow), we used non-modified DNA-oligonucleotides (synthesized byEuroGentec, Belgium). For the actual transfection experiments in musclecells, we used 2′-O-methyl-phosphorothioate oligoribonucleotides (alsosynthesized by EuroGentec, Belgium). These modified RNA oligonucleotidesare known to be resistant to endonucleases and RNaseH, and to bind toRNA with high affinity. The sequences of those AONs that were eventuallyeffective and applied in muscle cells in vitro are shown below. Thecorresponding mouse and human-specific AONs are highly homologous butnot completely identical.

The listing below refers to the deoxy-form used for testing, in thefinally used 2-O-methyl ribonucleotides all T's should be read as U's.

mAON#2: (SEQ ID NO: 1) 5′ GCAATGTTATCTGCTT mAON#3: (SEQ ID NO: 2) 5′GTTATCTGCTTCTTCC mAON#4: (SEQ ID NO: 3) 5′ CTGCTTCTTCCAGCC mAON#5:(SEQ ID NO: 4) 5′ TCTGCTTCTTCCAGC mAON#6: (SEQ ID NO: 5) 5′GTTATCTGCTTCTTCCAGCC mAON#7: (SEQ ID NO: 6) 5′ CTTTTAGCTGCTGCTC mAON#8:(SEQ ID NO: 7) 5′ GTTGTTCTTTTAGCTGCTGC mAON#9: (SEQ ID NO: 8) 5′TTAGCTGCTGCTCAT mAON#10: (SEQ ID NO: 9) 5′ TTTAGCTGCTGCTCATCTCC mAON#11:(SEQ ID NO: 10) 5′ CTGCTGCTCATCTCC hAON#4: (SEQ ID NO: 11) 5′CTGCTTCCTCCAACC hAON#6: (SEQ ID NO: 12) 5′ GTTATCTGCTTCCTCCAACC hAON#8:(SEQ ID NO: 13) 5′ GCTTTTCTTTTAGTTGCTGC hAON#9: (SEQ ID NO: 14) 5′TTAGTTGCTGCTCTT hAON#11: (SEQ ID NO: 15) 5′ TTGCTGCTCTTTTCC

Gel Mobility Shift Assays

The efficacy of the AONs is determined by their binding affinity for thetarget sequence. Notwithstanding recent improvements in computersimulation programs for the prediction of RNA-folding, it is difficultto speculate which of the designed AONs would be capable of binding thetarget sequence with a relatively high affinity. Therefore, we performedgel mobility shift assays (according to protocols described by Bruice etal., 1997). The exon 46 target RNA fragment was generated by in vitroT7-transcription from a PCR fragment (amplified from either murine orhuman muscle mRNA using a sense primer that contains the T7 promotersequence) in the presence of 32P-CTP. The binding affinity of theindividual AONs (0.5 pmol) for the target transcript fragments wasdetermined by hybridization at 37° C. for 30 minutes and subsequentpolyacrylamide (8%) gel electrophoresis. We performed these assays forthe screening of both the mouse and human-specific AONs (FIG. 3). Atleast 5 different mouse-specific AONs (mAON#4, 6, 8, 9 and 11) and fourcorresponding human-specific AONs (hAON#4, 6, 8, and 9) generated amobility shift, demonstrating their binding affinity for the target RNA.

Transfection into Muscle Cell Cultures

The exon 46-specific AONs which showed the highest target bindingaffinity in gel mobility shift assays were selected for analysis oftheir efficacy in inducing the skipping in muscle cells in vitro. In alltransfection experiments, we included a non-specific AON as a negativecontrol for the specific skipping of exon 46. As mentioned, the systemwas first set up in mouse muscle cells. We used both proliferatingmyoblasts and post-mitotic myotube cultures (expressing higher levels ofdystrophin) derived from the mouse muscle cell line C2C12. For thesubsequent experiments in human-derived muscle cell cultures, we usedprimary muscle cell cultures isolated from muscle biopsies from oneunaffected individual and two unrelated DMD patients carrying a deletionof exon 45. These heterogeneous cultures contained approximately 20-40%myogenic cells. The different AONs (at a concentration of 1 μM) weretransfected into the cells using the cationic polymer PEI (MBIFermentas) at a ratio-equivalent of 3. The AONs transfected in theseexperiments contained a 5′ fluorescein group which allowed us todetermine the transfection efficiencies by counting the number offluorescent nuclei. Typically, more than 60% of cells showed specificnuclear uptake of the AONs. To facilitate RT-PCR analysis, RNA wasisolated 24 hours post-transfection using RNAzol B (CamPro Scientific,The Netherlands).

RT-PCR and Sequence Analysis

RNA was reverse transcribed using C. therm. polymerase (Roche) and anexon 48-specific reverse primer. To facilitate the detection of skippingof dystrophin exon 46, the cDNA was amplified by two rounds of PCR,including a nested amplification using primers in exons 44 and 47 (forthe human system), or exons 45 and 47 (for the mouse system). In themouse myoblast and myotube cell cultures, we detected a truncatedproduct of which the size corresponded to exon 45 directly spliced toexon 47 (FIG. 4). Subsequent sequence analysis confirmed the specificskipping of exon 46 from these mouse dystrophin transcripts. Theefficiency of exon skipping was different for the individual AONs, withmAON#4 and #11 showing the highest efficiencies. Following thesepromising results, we focused on inducing a similar modulation ofdystrophin splicing in the human-derived muscle cell cultures.Accordingly, we detected a truncated product in the control musclecells, corresponding to exon 45 spliced to exon 47. Interestingly, inthe patient-derived muscle cells, a shorter fragment was detected, whichconsisted of exon 44 spliced to exon 47. The specific skipping of exon46 from the human dystrophin transcripts was confirmed by sequence data.This splicing modulation of both the mouse and human dystrophintranscript was neither observed in non-transfected cell cultures nor incultures transfected with a non-specific AON.

Immunohistochemical Analysis

We intended to induce the skipping of exon 46 in muscle cells frompatients carrying an exon 45 deletion in order to restore thetranslation and synthesis of a dystrophin protein. To detect adystrophin product upon transfection with hAON#8, the twopatient-derived muscle cell cultures were subject to immunocytochemistryusing two different dystrophin monoclonal antibodies (Mandys-1 andDys-2) raised against domains of the dystrophin protein located proximaland distal of the targeted region respectively. Fluorescent analysisrevealed restoration of dystrophin synthesis in both patient-derivedcell cultures (FIG. 5). Approximately at least 80% of the fibers stainedpositive for dystrophin in the treated samples.

Our results show, for the first time, the restoration of dystrophinsynthesis from the endogenous DMD gene in muscle cells from DMDpatients. This is a proof of principle of the feasibility of targetedmodulation of dystrophin pre-mRNA splicing for therapeutic purposes.

Targeted Skipping of Exon 51 Simultaneous Skipping of Dystrophin Exons

The targeted skipping of exon 51. We demonstrated the feasibility ofAON-mediated modulation of dystrophin exon 46 splicing, in mouse andhuman muscle cells in vitro. These findings warranted further studies toevaluate AONs as therapeutic agents for DMD. Since most DMD-causingdeletions are clustered in two mutation hot spots, the targeted skippingof one particular exon can restore the reading frame in series ofpatients with different mutations (see Table 1). Exon 51 is aninteresting target exon. The skipping of this exon is therapeuticallyapplicable in patients carrying deletions spanning exon 50, exons 45-50,exons 48-50, exons 49-50, exon 52, and exons 52-63, which includes atotal of 15% of patients from our Leiden database.

We designed a series of ten human-specific AONs (hAON#21-30, see below)directed at different purine-rich regions within dystrophin exon 51.These purine-rich stretches suggested the presence of a putative exonsplicing regulatory element that we aimed to block in order to inducethe elimination of that exon during the splicing process. Allexperiments were performed according to protocols as described for theskipping of exon 46 (see above). Gel mobility shift assays wereperformed to identify those hAONs with high binding affinity for thetarget RNA. We selected the five hAONs that showed the highest affinity.These hAONs were transfected into human control muscle cell cultures inorder to test the feasibility of skipping exon 51 in vitro. RNA wasisolated 24 hours post-transfection, and cDNA was generated using anexon 53- or 65-specific reverse primer. PCR-amplification of thetargeted region was performed using different primer combinationsflanking exon 51. The RT-PCR and sequence analysis revealed that we wereable to induce the specific skipping of exon 51 from the humandystrophin transcript. We subsequently transfected two hAONs (#23 and#29) shown to be capable of inducing skipping of the exon into sixdifferent muscle cell cultures derived from DMD-patients carrying one ofthe mutations mentioned above. The skipping of exon 51 in these cultureswas confirmed by RT-PCR and sequence analysis (FIG. 7). Moreimportantly, immunohistochemical analysis, using multiple antibodiesraised against different parts of the dystrophin protein, showed in allcases that, due to the skipping of exon 51, the synthesis of adystrophin protein was restored.

Exon 51-specific hAONs:

hAON#21: (SEQ ID NO: 16) 5′ CCACAGGTTGTGTCACCAG hAON#22: (SEQ ID NO: 17)5′ TTTCCTTAGTAACCACAGGTT hAON#23: (SEQ ID NO: 18) 5′ TGGCATTTCTAGTTTGGhAON#24: (SEQ ID NO: 19) 5′ CCAGAGCAGGTACCTCCAACATC hAON#25:(SEQ ID NO: 20) 5′ GGTAAGTTCTGTCCAAGCCC hAON#26: (SEQ ID NO: 21) 5′TCACCCTCTGTGATTTTAT hAON#27: (SEQ ID NO: 22) 5′ CCCTCTGTGATTTT hAON#28:(SEQ ID NO: 23) 5′ TCACCCACCATCACCCT hAON#29: (SEQ ID NO: 24) 5′TGATATCCTCAAGGTCACCC hAON#30: (SEQ ID NO: 25) 5′ CTGCTTGATGATCATCTCGTT

Simultaneous Skipping of Multiple Dystrophin Exons

The skipping of one additional exon, such as exon 46 or exon 51,restores the reading frame for a considerable number of different DMDmutations. The range of mutations for which this strategy is applicablecan be enlarged by the simultaneous skipping of more than one exon. Forinstance, in DMD patients with a deletion of exon 46 to exon 50, onlythe skipping of both the deletion-flanking exons 45 and 51 enables thereestablishment of the translational reading frame.

ERS-Independent Exon Skipping

A mutation in exon 29 leads to the skipping of this exon in two Beckermuscular dystrophy patients (Ginjaar at al., 2000, EJHG, vol. 8, p.793-796). We studied the feasibility of directing the skipping of exon29 through targeting the site of mutation by AONs. The mutation islocated in a purine-rich stretch that could be associated with ERSactivity. We designed a series of AONs (see below) directed to sequencesboth within (h29AON#1 to h29AON#6) and outside (h29AON#7 to h29AON#11)the hypothesized ERS. Gel mobility shift assays were performed (asdescribed) to identify those AONs with highest affinity for the targetRNA (FIG. 8). Subsequently, h29AON#1, #2, #4, #6, #9, #10, and #11 weretransfected into human control myotube cultures using the PEItransfection reagent. RNA was isolated 24 hrs post-transfection, andcDNA was generated using an exon 31-specific reverse primer.PCR-amplification of the targeted region was performed using differentprimer combinations flanking exon 29. This RT-PCR and subsequentsequence analysis (FIGS. 8B and 8C) revealed that we were able to inducethe skipping of exon 29 from the human dystrophin transcript. However,the AONs that facilitated this skipping were directed to sequences bothwithin and outside the hypothesized ERS (h29AON#1, #2, #4, #6, #9, and#11). These results suggest that skipping of exon 29 occurs independentof whether or not exon 29 contains an ERS and that, therefore, thebinding of the AONs to exon 29 more likely inactivated an exon inclusionsignal rather than an ERS. This proof of ERS-independent exon skippingmay extend the overall applicability of this therapy to exons withoutERS's.

h29AON#1: (SEQ ID NO: 26) 5′ TATCCTCTGAATGTCGCATC h29AON#2:(SEQ ID NO: 27) 5′ GGTTATCCTCTGAATGTCGC h29AON#3: (SEQ ID NO: 28) 5′TCTGTTAGGGTCTGTGCC h29AON#4: (SEQ ID NO: 29) 5′ CCATCTGTTAGGGTCTGTGh29AON#5: (SEQ ID NO: 30) 5′ GTCTGTGCCAATATGCG h29AON#6: (SEQ ID NO: 31)5′ TCTGTGCCAATATGCGAATC h29AON#7: (SEQ ID NO: 32) 5′ TGTCTCAAGTTCCTCh29AON#8: (SEQ ID NO: 33) 5′ GAATTAAATGTCTCAAGTTC h29AON#9:(SEQ ID NO: 34) 5′ TTAAATGTCTCAAGTTCC h29AON#10: (SEQ ID NO: 35) 5′GTAGTTCCCTCCAACG h29A0N#11: (SEQ ID NO: 36) 5′ CATGTAGTTCCCTCCAON-induced exon 46 skipping in vivo in murine muscle tissue.

Following the promising results in cultured muscle cells, we tested thedifferent mouse dystrophin exon 46-specific AONs in vivo by injectingthem, linked to polyethylenimine (PEI), into the gastrocnemius musclesof control mice. With mAON#4, #6, and #11, previously shown to beeffective in mouse muscle cells in vitro, we were able to induce theskipping of exon 46 in muscle tissue in vivo as determined by bothRT-PCR and sequence analysis (FIG. 9). The in vivo exon 46 skipping wasdose-dependent with highest efficiencies (up to 10%) following injectionof 20 μg per muscle per day for two subsequent days.

REFERENCES

Achsel et al., 1996, J. Biochem. 120, pp. 53-60.

Bruice T. W. and Lima, W. F., 1997, Biochemistry 36(16): pp. 5004-5019.

Brunak at al., 1991, J. Mol. Biol. 220, pp. 49-65.

Dunckley, M. G. et al., 1998, Human molecular genetics 7, pp. 1083-1090.

Ginjaar et al., 2000, EJHG, vol. 8, pp. 793-796.

Mann et al., 2001, PNAS vol. 98, pp. 42-47.

Tanaka et al., 1994 Mol. Cell. Biol. 14, pp. 1347-1354.

Wilton, S. D., et al., 1999, Neuromuscular disorders 9, pp. 330-338.

Details and background on Duchenne Muscular Dystrophy and relateddiseases can be found on website http://www.dmd.nl

1. An isolated antisense oligonucleotide of 14 to 40 nucleotides comprising a part of hAON#23 (5′-TGGCATTTCTAGTTTGG-3′ (SEQ ID NO: 18)),wherein said oligonucleotide induces skipping of exon 51 of the dystrophin pre-mRNA in a cell, said oligonucleotide comprising a modification for increasing its resistance to an endonuclease in a cell.
 2. An isolated antisense oligonucleotide of 14 to 40 nucleotides comprising a part of hAON#24 (5′-CCAGAGCAGGTACCTCCAACATC-3′ (SEQ ID NO:19)), wherein said oligonucleotide induces skipping of exon 51 of the dystrophin pre-mRNA in a cell, said oligonucleotide comprising a modification for increasing its resistance to an endonuclease in a cell.
 3. An isolated antisense oligonucleotide of 14 to 40 nucleotides being complementary to a part of exon 51 and comprising a part of hAON#23 (5′-TGGCATTTCTAGTTTGG-3′ (SEQ ID NO: 18)), and a part of hAON#24 (5′-CCAGAGCAGGTACCTCCAACATC-3′ (SEQ ID NO:19)), wherein said oligonucleotide induces skipping of exon 51 of the dystrophin pre-mRNA in a cell, and said oligonucleotide comprising a modification for increasing its resistance to an endonuclease in a cell.
 4. The oligonucleotide of claim 1, comprising complementarity to a part of exon
 51. 5. The oligonucleotide of claim 1, wherein said oligonucleotide interferes with an exon recognition sequence.
 6. The oligonucleotide of claim 1, wherein said oligonucleotide comprises a sequence complementary to an exon recognition sequence.
 7. The oligonucleotide of claim 1, wherein the cell is muscle cell.
 8. The oligonucleotide of claim 1, wherein the endonuclease is RNAse.
 9. The oligonucleotide of claim 1, which is a Peptide Nucleic Acid (PNA).
 10. The oligonucleotide of claim 1, wherein each T has been replaced by a U.
 11. The oligonucleotide of claim 10, wherein said oligonucleotide is a 2′-O-methyl oligoribonucleotide.
 12. The oligonucleotide of claim 11, wherein said oligonucleotide is a 2′-O-methyl phosphorothioate oligoribonucleotide.
 13. A nucleic acid delivery vehicle comprising a transcription unit expressing an antisense oligonucleotide consisting of 14 to 40 nucleotides, said oligonucleotide comprising a part of hAON#23 (5′ TGGCATTTCTAGTTTGG (SEQ ID NO: 18)), wherein said oligonucleotide induces skipping of exon 51 of the dystrophin pre-mRNA in a cell.
 14. A nucleic acid delivery vehicle comprising a transcription unit expressing an antisense oligonucleotide consisting of 14 to 40 nucleotides, comprising a part of hAON#24 (5′-CCAGAGCAGGTACCTCCAACATC-3′ (SEQ ID NO:19)), wherein said oligonucleotide induces the skipping of exon 51 of the dystrophin pre-mRNA in a cell.
 15. A nucleic acid delivery vehicle comprising a transcription unit expressing an antisense oligonucleotide consisting of 14 to 40 nucleotides, comprising a part of hAON#23: 5′ TGGCATTTCTAGTTTGG (SEQ ID NO: 18) and a part of hAON#24:: 5′-CCAGAGCAGGTACCTCCAACATC-3′ (SEQ ID NO:19), wherein said oligonucleotide induces skipping of exon 51 of the dystrophin pre-mRNA in a cell.
 16. A composition comprising the oligonucleotide of claim
 1. 17. A composition comprising the nucleic acid delivery vehicle of claim
 13. 18. A pharmaceutical formulation comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable vehicle.
 19. A pharmaceutical formulation comprising the nucleic acid delivery vehicle of claim 13 and a pharmaceutically acceptable vehicle.
 20. A method for directing splicing of a dystrophin pre-mRNA in a cell by inducing the skipping of exon 51 of the dystrophin pre-mRNA to produce a dystrophin protein comprising: providing the cell with an antisense oligonucleotide of claim 1; and wherein said oligonucleotide induces skipping of exon 51 from said dystrophin pre-mRNA in said cell.
 21. The oligonucleotide of claim 1, said oligonucleotide being 17 to 40 nucleotides in length.
 22. The oligonucleotide of claim 1, said oligonucleotide being 14 to 25 nucleotides in length.
 23. The nucleic acid delivery vehicle of claim 13, said oligonucleotide being 17 to 40 nucleotides in length.
 24. The nucleic acid delivery vehicle of claim 13, said oligonucleotide being 14 to 25 nucleotides in length.
 25. A method for treating Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) in a patient by inducing the skipping of exon 51 of the human dystrophin pre-mRNA, said method comprising providing the oligonucleotide of claim 1 to said patient, wherein said oligonucleotide induces skipping of said exon in said patient thereby treating DMD or BMD. 